铜绿假单胞菌重组Bb-OprI疫苗诱导小鼠保护力和脾细胞因子基因表达变化的研究

Study on protection by and changes in expression of cytokine genes in splenocytes from mice inoculated with a recombinant Bb-OprI vaccine against Pseudomonas aeruginosa

目的研究铜绿假单胞菌重组Bb-OprI疫苗免疫及PA01株攻击后,小鼠肺组织细菌菌落计数和脾细胞因子IL-2、IFN-γ和IL-12基因表达的变化。方法取5×109个CFU Bb-OprI疫苗灌胃接种BALB/c鼠,每周3次,连续接种3周。首次免疫后4周,用5×106个CFU的PA01株滴鼻攻击。攻击后2周处死小鼠,取肺脏,经常规培养后计数菌落数;取小鼠脾脏,制备脾细胞悬液,用PaAg刺激培养,48h后收集脾细胞,PCR扩增IL-2、IFN-γ和IL-12基因。结果Bb-OprI疫苗组、空载体对照组和Bb对照组小鼠肺组织菌落数分别为(0.46±0.09)×10~8 CFU、(5.42±0.79)×10~8 CFU和(6.20±0.95)×10~8 CFU,差异有统计学意义(P<0.01);疫苗组小鼠脾细胞扩增出453bp的IL-2基因、399bp的IFN-γ基因和300bp的IL-12基因,两对照组PCR扩增均阴性。结论重组Bb-OprI疫苗可诱导小鼠脾细胞因子IL-2、IFN-γ和IL-12基因表达升高,产生Th1型免疫应答从而抵抗Pa的感染。

Objective To study the number of pulmonary bacterial colonies and changes in expression of genes coding for the cytokines IL-2,IFN-γ,and IL-12 in splenocytes from mice immunized with a recombinant Bb-OprI vaccine against Pseudomonas aeruginosa and challenged with the PA01 strain. Methods BALB/c mice were intragastrically administered 5×109 CFU of the Bb-OprI vaccine 3 times a week for 3 weeks,and they were challenged intranasally with 5×106 CFU of the PA01 strain 4 weeks after the first immunization.Two weeks after the challenge,all mice were sacrificed.The lungs were removed,and the number of pulmonary bacterial colonies was counted after routine culture.The spleen was removed to prepare a suspension of splenocytes that were stimulated with PaAg during 48 h of culturing.The splenocytes were collected and then the IL-2,IFN-γand IL-12 genes were amplified using PCR. Results The number of pulmonary bacterial colonies was（0.46±0.09）×10-8 CFU in the Bb-OprI vaccine group,（5.42±0.79）×10-8 CFU in the vector control group,and（6.20±0.95）×108 CFU in the Bb control group.The number of pulmonary bacterial colonies differed significantly（P〈0.01）among the three groups.The IL-2 gene（453 bp）,the IFN-γgene（399 bp）,and the IL-12 gene（300 bp）were amplified from splenocytes from mice in the vaccine group but not from splenocytes from the two control groups. Conclusion A recombinant Bb-OprI vaccine may cause an increase in expression of genes coding for the cytokines IL-2,IFN-γ,and IL-12 in splenocytes,and it may cause a Th1 immune response to a P.aeruginosainfection.